Formation of 4-hydroxy-2-nonenal-modified proteins and 3-nitro-L-tyrosine in rat island skin flaps during and after ischemia.

4-Hydroxy-2-nonenal (HNE)-modified proteins and 3-nitro-L-tyrosine were evaluated as a specific marker of reactive oxygen species (ROS)- and nitric oxide (NO)-mediated peroxynitrite-induced tissue injuries in ischemic and reperfused skin flap by Western blot analysis. Specimens were taken from island skin flaps of rats during the following three conditions: ischemia only, 5 hours of ischemia and reperfusion, and 10 hours of ischemia and reperfusion. HNE-modified proteins and 3-nitro-L-tyrosine increased with ischemic time (3, 6, and 10 hours postischemia). In the reperfused skin flap after both 5 and 10 hours of ischemia, HNE-modified proteins and 3-nitro-L-tyrosine were increased 3 hours postreperfusion, and they reached a maximum 6 hours after reperfusion. HNE-modified proteins and 3-nitro-L-tyrosine 1 hour postreperfusion were higher with 10 hours ischemia-reperfusion than with 5 hours ischemia-reperfusion. These results indicate (1) that ROS- and NO-induced peroxynitrite-mediated cytotoxicity in ischemic flaps is dependent on the ischemic period and (2) that ROS- and NO-induced peroxynitrite-mediated cytotoxicity occurs during an early stage of reperfusion if the ischemic period is long.